Enhanced CD103 Expression and Reduced Frequencies of Virus-Specific CD8+ T Cells Among Airway Lymphocytes After Influenza Vaccination of Mice Deficient in Vitamins A + D

Abstract

Previous research has evaluated antibody responses toward an influenza virus vaccine in the context of deficiencies for vitamins A and D (VAD+VDD). Results showed that antibodies and antibody-forming cells in the respiratory tract were reduced in VAD+VDD mice. However, effectors were recovered when oral supplements of vitamins A + D were delivered at the time of vaccination. Here we address the question of how vaccine-induced CD8+ T cell responses are affected by deficiencies for vitamins A + D. VAD+VDD and control mice were vaccinated with an intranasal, cold-adapted influenza virus A/Puerto Rico/8/34 vaccine, with or without oral supplements of vitamins A + D. Results showed that the percentages of vaccine-induced CD8+ T cell and total CD4+ T cell responses were low among lymphocytes in the airways of VAD+VDD animals compared to controls. The CD103 membrane marker, a protein that binds e-cadherin (expressed on respiratory tract epithelial cells), was unusually high on virus-specific T cells in VAD+VDD mice compared to controls. Interestingly, when T cells specific for the PA224-233/Db epitope were compared with T cells specific for the NP366-374/Db epitope, the former population was more strongly positive for CD103. Preliminary experiments revealed normal or above-normal percentages for vaccine-induced T cells in airways when VAD+VDD animals were supplemented with vitamins A + D at the time of vaccination and on days 3 and 7 after vaccination. Our results suggest that close attention should be paid to levels of vitamins A and D among vaccine recipients in the clinical arena, as low vitamin levels may render individuals poorly responsive to vaccines.

Keywords: bronchoalveolar lavage; influenza virus; vaccine-induced T cells; vitamin deficiency; vitamins A and D.

Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.

Vaccine-specific T cell responses among VAD+VDD and control mice. Flow cytometry profiles (A–J) are shown for an experiment in which BAL samples were pooled within groups of mice after mice received a cold-adapted influenza virus vaccine. Day 10, lymphocyte-gated (based on FSC, SSC) BAL profiles are shown for control mice (Top row), VAD+VDD mice (second row, “Double”), VAD+VDD mice given a vitamin D supplement (third row, “Double + D”), VAD+VDD mice given a vitamin A supplement (fourth row, “Double + A”), or VAD+VDD mice given a supplement of vitamins A + D (fifth row, “Double + A+D”). In the left column, NP366–374/Db Tet+ staining is shown on the y axis and CD8+ staining is shown on the x axis. In the right column, a histogram is shown for CD4 staining. As demonstrated, VAD+VDD animals exhibited low percentages of CD8+ NP366–374/Db Tet+ and CD4+ cells compared with control animals among lymphocytes (red circles), but responses matched or exceeded normal levels when animals were supplemented with vitamins A+D. In this experiment, there were five mice per group, and samples were mixed within each group before assay. Total cell counts based on flow cytometry were 2.5 × 106 and 3.1 × 106 for VAD+VDD and control animals, respectively. BAL, bronchoalveolar lavage. Color images available online at www.liebertpub.com/vim

FIG. 2.

Reduced virus-specific CD8+ and CD4+ cell frequencies among airway lymphocytes in vaccinated VAD+VDD animals. Individual animals were tested in two separate experiments. Results for these combined experiments are shown to demonstrate differences between VAD+VDD animals and control animals for the percentages of NP366–374/Db Tet+ CD8+ cells (A), the percentages of PA224–233/Db Tet+ CD8+ cells (B), and the percentages of CD4+ cells (C) among airway lymphocytes in the control and VAD+VDD (“Double”) groups.

FIG. 3.

CD44+ and CD11a+ cell frequencies among CD8+ cells in VAD+VDD and control mice. The phenotypes of CD8+ cells are shown with respect to membrane markers CD44 and CD11a. (A–E) illustrate the frequencies of CD44+ cells among CD8+ cells and (F–J) illustrate the frequencies of CD11a+ cells among CD8+ cells.

FIG. 4.

CD103Hi cell frequencies among CD8+ cells in VAD+VDD and control animals. The percentages of CD103Hi cells among CD8+ cells are shown (A–E). Also shown are percentages for individual mice, comparing CD103Hi cells among CD8+ cells bound by the NP366–374/Db tetramer (F) or the PA224–233/Db tetramer (G) in VAD+VDD mice compared with controls. In (H) are shown percentages for CD103Hi cells among CD8+ cells (second column), among NP366–374/Db Tet+ cells (third column), or among PA224–233/Db Tet+ cells (fourth column) in an experiment with five different mouse groups. Percentages of CD103Hi cells among CD8+ cells in VAD+VDD mice were higher than in control mice in each of four experiments (see red circle in panel B, and panel F for examples), and dropped in each of two experiments when animals were supplemented with vitamins A+D (E). CD103Hi percentages were also higher among PA224–233/Db-specific cells than among NP366–374/Db-specific cells in each of four experiments. Color images available online at www.liebertpub.com/vim